Diagnostic reagents for the determination of gamma-glutamyl transpeptidase in human serum

ABSTRACT

DIAGNOSTIC REAGENTS FOR THE DETERMINATION OF Y-GLUTAMYL TRANSPEPTIDASE IN HUMAN SERUM WHICH COMPRISES (1) Y-LGLUTAMIC ACID P-NITROANILIDE, (2) ONE OR MORE SURFACE ACTIVE AGENTS SELECTED FROM THE GROUP CONSISTING OF AMPHOTERIC SURFACE ACTIVE AGENTS AND NON-IONIC SURFACE ACTIVE AGENTS AND (3) A BUFFER SOLUTION SELECTED FROM THE GROUP CONSISTING OF COLLIDINE-, BORIC ACID-, VERONAL (5,5-DIETHYLBARBITURIC ACID)- AND AMMONIA-BUFFER SOLUTIONS. THE REAGENTS OF THIS INVENTION ARE VERY STABLE AT A HIGH CONCENTRATION AND PRACTICALLY USEFUL FOR THE DIAGNOSIS OF VARIOUS TYPES OF LIVER DISEASES, E.G. CANCER OF LIVER, OBSTRUCTIVE LIVER DISEASES AND THE LIKE.

United States Patent DIAGNOSTIC REAGENTS FOR THE DETERMINA- TION 0F 'y-GLUTAMYL TRANSPEPTIDASE IN HUMAN SERUM Kazuo Nakanishi and Shuzo Takei, Tokyo, Japan, assignors to Sankyo Company Limited, Tokyo, Japan No Drawing. Filed June 17, 1970, Ser. No. 47,141

Claims priority, appliizatiolggapan, June 23, 1969,

Int. Cl. c1zk 1/04 US. Cl. 195-99 5 Claims ABSTRACT OF THE DISCLOSURE This invention relates to a diagnostic reagent. More particularly, it is concerned with a diagnostic reagent for the detection and quantitative determination of 'y-glutamyl transpeptidase (hereinafter referred to as 'y-G'IPase) in human serum which comprises a specific combination of 'y-L-glutamic acid p-nitroanilide, a selected surface active agent and a selected buffer solution.

It is known in the art that 'y-GTPase is predominantly found in liver and kidney and also that the higher 'y- GTPase activity in serum is seen in those patients with liver diseases, e.g. cancer of liver, obstructive liver diseases and the like.

Recently, it was reported'by A. Szewczuk et al. [Clinica Chimica Acta, 7, 755, 1962], S. Lukasik et al. [Schweizeriche Medizinische Wochenschrift, 98, 81, 1968] and M. Orlowski et a1. [Clinica Chemica Acta, 15, 387, 1967] that a 'y-GTPase method is very much more sensitive than the alkaline phosphatase method [Journal of Clinical Pathology, 7, 322, 1954] and the leucine aminopeptidase method [Cancer, 11, 283, 1958], which have been utilized for the diagnosis of liver diseases.

Thus, it is earnestly desired and attempted in the art to develop the above-mentioned 'y-GTPase method to a practically utilizable one, for instance, in order to adopt such a method in a hospital as a routine examination procedure. However, favourable success has not been attained because of poor solubility of -L-glutamic acid p-nitroanilide (hereinafter referred to as 'y-GNA), which is the most suitable substrate for the determination of the 'y-GTPase activity, and of instability of a solution of this substrate. Thus, this determination method particularly involves determining the 'y-GTP'ase activity in the presence of a substrate at a concentration of 5 mM. in a 0.1 M tris [tris (hydroxymethyl)aminomethane] buffer solution of pH 8 at 37 C. However, such a prior art method has some drawbacks in that (1), as the substrate, 'y-GNA, tends to be decomposed into glutamic acid and p-nitroaniline in a tris butfer solution of pH 7-9, i.e. the ontimum pH range, the preparation of the reagent and the determination procedure should be done within a short period of time and, if not, it leads to low accuracy of the measured value and (2), as the solubility of the substrate in the above tris buffer solution within the abovespecified pH range is at most about 2 mM. at ordinary temperature, an unstable supersaturated solution of the substrate, which is prepared by dissolution with heating and chilling, should be employed in order to obtain the solution having a concentration of about 5 mM. for the determination of the -GTPase.

As a result of our extensive studies on an improved and more practical diagnostic reagent for the determination of 'y-GTPase in human serum, it has now been found that a 'y-GNA solution, which is stable at a high concentration and is useful as a diagnostic reagent, can be obtained by selecting and employing a particular combination of 'y-GNA with a specific surface active agent and a specific buffer solution.

It is, accordingly, a primary object of this invention to provide a highly stable and high substrate concentration diagnostic reagent for the determination of 'y-GTPase in human serum which comprises a combination of 'y-GNA and a particular amphoteric or non-ionic surface active agent and a particular buffer solution.

Other objects and advantages of this invention will be apparent to those skilled in the art from the following detailed description of this invention.

As outlined hereinabove, the diagnostic reagent of this invention has been completed based upon the above finding and, in particular, comprises a novel combination of (2) one or more surface active agents selected from the group consisting of amphoteric surface active agents and non-ionic surface active agents, and

(3) a buffer solution selected from the group consisting of a collidine buffer solution, a boric acid buffer solution, a 5,5-diethylbarbituric acid buffer solution and an ammonia buifer solution.

The procedure or manner wherein the above finding has been led will be more illustratively stated hereinbelow.

First, studies on the solubility of the substrate in a wide variety of solvents were made as follows. Where a conventional solvent was employed, for example, an alcohol, e.g. methanol, ethanol, propanol, butanol, glycerol and the like; a ketone, e.g. acetone, methyl ethyl ketone, and the like; a lower aliphatic acid ester, e.g. ethyl acetate, butyl acetate and the like; a heterocyclic base, e.g. pyridine, quinoline and the like; an aromatic hydrocarbon, e.g. benzene, toluene and the like; dimethylformamide; water; a mixture thereof, there could be only obtained a solution of the substrate having a concentration below 2 mM. in each of the above-listed solvents at a temperature employed for the determination in an enzyme reaction, and, Where employed a 30% aqueous dirnethyl sulfoxide solution, there could also be obtained only a 3.5 mM. solution of the substrate. In short, the solution of the required concentration could not be obtained with any of the above-listed solvents.

If various surface active agents are employed together, the substrate solution of a high concentration above approximately 5 mM. could be provided. Thus, the following surface active agents were tested: For instance, cationic surface active agents such as those containing quarternary ammonium ions, e.g. benzethonium chloride, benzalkonium chloride and the like; anionic surface active agents such as those containing derivatives of carboxylic, sulfonic or sulfuric acids, e.g. sodium laurate, sodium dodecylbenzenesulfonate, sodium alcohol polyethenoxy sulfate; amphoteric surface active agents such as those containing imidazoline compounds, e.g. Miranol, or N-acyl sarcosine, e.g. sodium N-lauroylsarcosine; as well as nonionic surface active agents such as those containing a mixed ether of an alcohol and a phenol, e.g. polyoxyethylene nonyl phenol ether, polyoxyethylene octyl alcohol ether.

Further, the reaction employed for accomplishment of the purpose of this invention is an enzymatic reaction and thus one should, of course, select such a surface active agent that could not inhibit this reaction.

As a result of our studies on the effect of the abovementioned cationic, anionic, amphoteric and non-ionic surface active agents, it was found that amphoteric and non-ionic surface active agents do not in any way inhibit the 'y-GTPaSe activity, whereas cationic and anionic surface active agents remarkably inhibit said activity.

Some representative examples of the above studies are summarized in the following Table 1, wherein the state of the reaction after the lapse of 30 minutes from the initiation of the reaction is shown by change in absorbance at mill (AE410) U Hyamine 1622, trade name, available from Rohm & Haas Co.,

Nikkol. NE s 202, trade name, available from Nikko Chemicals Co., Ltd., Japan.

3 Nikkol Sarcosinate" LN, trade name, available from Nikko Chemicals Co., Ltd., Japan.

4 Miranol C 2 M -SF trade name, available from The Miranol Chemical Co. Incorporation, .S.A.

6 Nilrkol N P-9, trade name, available from Nikko Chemicals Co., b l i cil BO 20, trade name, available from Nikko Chemicals Co., Ltd., Japan.

The method for the determination of the -GTPase activity which is employed herein is summarized below, the disclosure of which is incorporated herein as a reference.

A solution of g. of one of the above-listed surface active agents, 150 mg. of 'y-GNA and 396 mg. of glycylglycine in about 90 ml. of distilled Water is adjusted to pH 8 with addition of hydrochloric acid or an aqueous solution of sodium hydroxide and made up the whole amount to 100 ml. with addition of a suitable amount of distilled water. The resulting solution is employed as a substrate solution. The mixture of 0.5 ml. of the substrate solution with 0.01 ml. of serum is allowed to stand at 37 C. for 30 minutes and then 2.5 ml. of an 1.3 M aqueous acetic acid solution is added thereto. The absorbance at 410 mp. is measured in a conventional manner. On the other hand, 0.5 ml. of the substrate solution is allowed to stand at 37 C. for 30 minutes and then 0.01 ml. of serum and 2.5 ml. of an 1.3 M aqueous acetic acid solution are added thereto. The absorbance at 410 mg is measured as above. The difference obtained by deducting the latter measured value from the former one is defined as the activity value, expressed as AE It is also found that the substrate, 7-GNA, which can be employed in this invention shows extremely diiferent stabilities in the same pH range, depending upon the kinds and types of the buffer solutions employed and that this substrate is very unstable in the tris buffer solution previously utilized for the determination of the -GTPase activity and various phosphate buffer solutions with a wide pH range from weakly acidic to weakly basic which are commonly utilized in an enzymatic reaction.

Some representative examples of the above studies are summarized in the following Table II, wherein the decomposition state of the substrate after the lapse of 3 days from the preparation of the test solution is shown by change in absorbance at 410 m, (M3

Tris (hydroxymethyl) aminomethane hydrochloric acid 6.0 Sodium phosphate-potassium phosphate 9.0

1 5,5-diethylbarbituric acid.

The method for the determination of the stability of the 'y-GNA in buffer solution which is employed herein is summarized below, the disclosure of which is incorporated herein as a reference.

In this method, the same substrate solution in the above Table I is employed except that Nikkol NP-9 is employed as a surface active agent and the indicated buffer solution of pH 8.0 is employed instead of the distilled water and the acid or base. Immediately and 3 days after the preparation, the absorbance at AE was measured. The difference obtained by deducting the former measured value from the latter one is defined as the stability value.

In a suitable embodiment of this invention, the final concentration of the surface active agent employed in this invention is desirably within the range of about 2.5 to 15% in the reagent for the determination of 'y-GTPase, with about 5% being most preferable. The ionic strength of the buifer solution employed in the present reagent may be widely varied mainly depending upon the kinds and types of the buffer solutions employed, but the preferable strength is within the range of about 0.05 to 0.3, with about 0.1 for the collidine or ammonia buffer solution, about 0.2 to 0.3 for the boric acid buffer solution and less than about 0.1 for the Veronal buffer solution being most preferable.

It is desirable in this invention to employ the concentration of the substrate, 'y-GNA, of about 4 to 6 mM., since accurate measured values can be constantly found.

Noticeable increase in the reaction rate may be accomplished, not essential, by further addition to the present reagent of a suitable amount of a peptide derivative having free terminal amino groups such as glycylglycine and the like as an acceptor for the 'y-glutamic acid which may be produced in situ as a consequence of an enzymatic reaction.

In quantitatively determining 'y-GTPase in human serum by means of the present diagnostic reagent, one may satisfactorily use the present diagnostic reagent prepared either previously or immediately before the application thereof.

The procedure for determining 'y-GTPase in human serum may be favourably carried out by a slight modification of the well-known determination method, for example, that disclosed by D. M. Dimov et al. in Clinica Chemica Acta, 16, 271 (1967). The details of the determination procedure will be set forth hereinbelow, the disclosure of which is incorporated herein as a reference.

(1) A mixture of 0.5 ml. of the reagent with 0.02 ml. of human serum is allowed to stand at 37 C. for minutes and then 2.5 H11. of a 1.3 M aqueous acetic acid solution is added thereto. The absorbance at 410 m is measured in a conventional manner. On the other hand, 0.5 ml. of the reagent is allowed to stand at 37 C. for 30 minutes and then 2.5 ml. of a 1.3 M aqueous acetic acid solution and 0.02 ml. of serum are added thereto. The absorbance at 410 m is also measured as above.

The difference obtained by deducting the latter measured value from the former one is defined as the activity value.

(2) Immediately after 3.0 ml. of the reagent is admixed with 0.02 ml. of human serum, the absorbance EXAMPLE 1 The diagnostic reagent was prepared having the following composition:

'y-GNA mg 150 A N hydrochloric acid ml.. 15 Glycylglycine m2 395 Nikkol NP-9 g M Veronal Sodium (sodium 5,5-diethylbarbiturate)sodium acetate (pH 8.0) ml 22 Distilled water (q.s.) to 100 ml.

The reagent was prepared as follows. The 'y-GNA was dissolved in the hydrochloric acid (referred to as Solution A). On the other hand, the glycylglycine and the Nikkol were dissolved in a portion of about 55 ml. of the distilled water and then the resulting solution adjusted to pH 8.0 by addition of dilute aqueous sodium hydroxide to form an alkaline solution (referred to as Solution B). To the Solution B were added the Solution A and the Veronal buffer solution and then a sufiicient amount of distilled water was added thereto to make up the whole volume to 100 ml.

EXAMPLE 2 The diagnostic reagent was prepared having the same composition as in the above Example 1 except that g. of Nikkol NP-9 was employed instead of the 5 g. and 30 ml. of /s M collidine-hydrochloric acid (pH 8.0) bufier solution was employed instead of the Veronal buffer solution.

EXAMPLE 3 The diagnostic reagent was prepared having the same composition as in the above Example 1 except that 2.5 g. of Nikkol NP-9 was employed instead of the 5 g. and 15.5 ml. of a ,5 N aqueous ammonia was employed instead of the Veronal buffer solution.

EXAMPLE 4 The diagnostic reagent was prepared having the same composition as in above Example 1 except that 18.3 ml. of /5 M boric acid-sodium carbonate buffer solution (pH 8.0) was employed instead of the Veronal buffer solution.

EXAMPLE 5 The diagnostic reagent was prepared having the same composition as in the above Example 1 except that 5 g. of

6 Nikkol BO 20 was employed instead of the Nikkol NP-9.

EXAMPLE 6 The diagnostic reagent was prepared having the same composition as in the above Example 1 except that 5 g. of Miranol C2M-SF was employed instead of the Nikkol NP-9.

EXAMPLE 7 The diagnostic reagent was prepared having the same composition as in the above Example 1 except that 5 g. of Miranol C2M-SF was employed instead of the Nikkol NP9 and 15.5 ml. of ,5 N aqueous ammonia was employed instead of the Veronal buffer solution.

What is claimed is:

1. A diagnostic reagent for the determination of 'yglutamyl transpeptidase in human serum which comprises a combination of (1) 'y-glutamic acid p-nitroanilide,

(2) one or more surface active agents selected from the group consisting of an N-acyl sarcosine, an imidazoline amphoteric compound, a polyoxyethylene alcohol ether and a polyoxyethylene phenol ether, and

(3) a buffer solution selected from the group consisting of a collidine buffer solution, a boric acid bufier solution, a 5,5-diethylbarbituric acid buifer solution and an ammonia buffer solution.

2. The reagent according to claim 1 wherein said surface active agent is that containing sodium lauroylsarcosine, an imidazoline, polyoxyethylene nonyl phenol ether or polyoxyethylene octyl alcohol ether.

3. The reagent according to claim 1 wherein said bufier solution is a collidine-hydrochloric acid buffer solution, a boric acid-sodium carbonate buffer solution, a sodium 5,S-diethylbarbiturate-sodium acetate buffer solution or aqueous ammonia.

4. The reagent according to claim 1 wherein the concentration of said surface active agent is 2.515% and said bufler solution has an ionic strength of 0.05-0.13.

5. The reagent according to claim 1 wherein it contains 0.15% of -L-glutamic acid p-nitroanilide, 5% of polyoxyethylene nonyl phenol ether and 22% of a M sodium 5,5-diethylbarbiturate-sodium acetate buffer solution.

References Cited UNITED STATES PATENTS ALVIN E. TANENHOLTZ, Primary Examiner M. D. HENSLEY, Assistant Examiner US. Cl. X.R. 

